Fast liquid chromatography–mass spectrometry glutathione measurement in whole blood: micromolar GSSG is a sample preparation artifact

Abstract

We describe a new, fast (6 min) and reliable method to measure reduced or oxidized glutathione (GSH) or (GSSG) in whole blood. The method is based on a LC/MS measurement in positive electrospray ionization mode after a chromatographic separation on a specific column which does not need any counter-ion in the mobile phase, improving the sensitivity of detection. A 50 μl sample of whole blood is sufficient for analysis. We demonstrate that the lack of an alkylating agent during the sample preparation brings out an underestimation of GSH and an artefactual production of GSSG, corresponding to 2–3% of GSH. The simultaneous use of N-ethyl-maleimide and a strong deproteinising acid prevents these two drawbacks. This efficient and new method of preparation and analysis lets us show that, unexpectedly, GSH is stable in whole blood for some hours and that deproteinised samples can be stored without GSH loss for at least three weeks at −20 or −80 °C. The reference interval, measured on 22 volunteers, on blood samples collected either with heparin or with EDTA, is 1310±118 μM for GSH and 0.62 μM for GSSG. The within-run precision of this method, with γ glutamyl-glutamic acid as an internal standard, evaluated in three successive series ( n=30), lies between 2.1 and 4.8% for a GSH level at 580 or 1150 μM. The one step sample preparation we propose seems well suited for GSH routine measurements in hospital laboratories and avoids any underestimation of GSH, a now well accepted biomarker of oxidative stress.